An emerging theme in structural biology is that disordered proteins play important roles in biological systems. For example, some disordered proteins adopt structure during important signaling or regulatory events. The structural determinants of these events, however, are unknown, precluding the correlation of structure and function. The cyclin-dependent kinase (Cdk) inhibitor, p21, is a regulatory target of two important tumor suppressors, p53 and BRCA1. In humans, p53-dependent tumor suppression involves activation of p21 that causes cell cycle arrest. We have shown that p21, and a related protein named p27, are dynamically disordered in solution. Despite this, p21 and p27 bind to cyclin/Cdk complexes, the timekeepers of the cell cycle, with high affinity and specificity. In the past, p21 and p27 were considered to be universal inhibitors of Cdks; recent studies, however, have shown that p21 and p27 inhibit only a subset of Cdks (i.e. Cdk2/cyclin A) and that they stabilize and activate others (i.e. Cdk4/cyclin D). One important aim of our studies is to uncover the physical basis for the dual functions of p21 and p27 through studies of protein structure and dynamics using NMR spectroscopy and studies of binding thermodynamics using isothermal titration calorimetry (ITC). The PI's laboratory has shown using NMR that p21 and p27 possess a transiently populated -helix in solution - the "linker helix" - and suggests that this structural feature is an important determinant of function. This hypothesis will be tested using protein engineering to both stabilize and destabilize the "linker helix" followed by the determination of binding parameters and activity. Future studies with p21 homologs will determine whether this structural feature is evolutionarily conserved. Also, NMR studies will be extended to p21 and p27 within cyclin/Cdk complexes to uncover the structural determinants of Cdk inhibition versus activation. Finally, ITC and other techniques are being used to elucidate the structural and thermodynamic basis for the specificity of p21 and p27 for cell cycle Cdks. This work is important because p21 and p27 regulate the cell growth arrest mechanism that is most often disrupted in human cancer and because the relationship between the structure and function of these proteins is not well understood.